MicroRNA-29a regulates the benzo[a]pyrene dihydrodiol epoxide-induced DNA damage response through Cdc7 kinase in lung cancer cells

Abstract

Cdc7 kinase is a key regulator of DNA replication and has an important role in the cellular DNA damage response by controlling checkpoint signaling and cell survival. Yet, how the activity of Cdc7 kinase is regulated is poorly understood. In silico analysis identified microRNA-29 (miR-29)-binding sites in the 3'-untranslated region (UTR) of both Cdc7 and its activating subunit Dbf4. We show that miR-29a binds to Cdc7 and Dbf4 3'-UTRs and regulates kinase levels. We find that in response to DNA damage, upregulation of Cdc7 kinase correlates with a downregulation in miR-29a. Enforced miR-29a expression prevents the accumulation of Cdc7 in response to the environmental genotoxin, benzo[a]pyrene dihydrodiol epoxide (BPDE) present in cigarette smoke, resulting in aberrant checkpoint signaling and increased cell lethality. As BPDE sensitivity was rescued by overexpression of miRNA-resistant Cdc7/Dbf4, we propose that Cdc7 kinase is an important target of miR-29a in determining cell survival from genotoxic stress caused by this environmental toxin.

Figure 1

miR-29a targets Cdc7 and Dbf4 mRNA at specific 3′-UTR sites. (a) Putative miR-29a-binding sites in the 3′-UTR regions of Cdc7 and Dbf4. (b) Luciferase reporter assay constructs containing the full-length 3′-UTR of Cdc7 (wt-Cdc7 3′-UTR) or the full-length 3′-UTR of Dbf4 (wt-Dbf4 3′-UTR) were used to generate stable cell lines expressing wt-Cdc7 3′-UTR luciferase or wt-Dbf4 3′-UTR luciferase. Stable cell lines were transfected with miR-ve or miR-29a, and luciferase activity measured. miR-29a overexpression caused a significant decrease in luciferase activity in both wt-Cdc7 3′-UTR (n=3, **P=0.015 ) and wt-Dbf4 3′-UTR (n=3, ***P=0.006) expressing cells compared with miR-ve. Values are presented as the mean±s.e.m. (c) Mutant constructs (Cdc7 mut 29-1 (sites 422–428 and 428–434), Cdc7 mut 29-2 (site 681–687), and the Dbf4 3′-UTR, Dbf4 mut 29 (site 497–503)) were used to generate stable cell lines, which were transfected with miR-29a or miR-ve and luciferase activity measured. miR-29a induced a significant decrease in luciferase activity in cells expressing wt-Cdc7 3′-UTR (n=5, ***P=5.7 × 10⁻⁶) and wt-Dbf4 3′-UTR (n=4, ***P=0.0001), but caused no significant difference in luciferase activity in cells expressing the mutant 3′-UTRs of Cdc7 or Dbf4.

Figure 2

miR-29a inhibits Cdc7 and Dbf4 expression. (a) MRC-5 and H1299 cells were transfected with miR-29a and mir-29a levels were determined by real-time quantitative PCR (RT–qPCR). (b) Western blot analysis was performed to determine Cdc7 and Dbf4 protein levels. β-Actin served as loading control. (c) Cells were reverse transfected with miR-ve or miR-29a. Forty-eight hours after transfection, cells were incubated with 10 μℳ 5-ethynl-2′-deoxyuridine for 1 h. Levels of 5-ethynl-2′-deoxyuridine incorporation were determined by the click reaction and fluorescence-activated cell sorting analysis.

Figure 3

miR-29a levels inversely correlate with Cdc7 and Dbf4 levels in response to BPDE. (a) A549 cells were treated with 100 or 600 nℳ BPDE for the indicated times and fractionated into CSK-soluble and CSK-insoluble fractions. Proteins were analyzed by SDS–polyacrylamide gel electrophoresis and immunoblotting. (b) RNA was also prepared and miR-29a levels were determined by real-time quantitative PCR (RT–qPCR). (c) A549 cells were treated with 10J UV. RNA and protein extracts were prepared at the indicated times. miR-29a and Cdc7 levels were determined by RT–qPCR and western blot, respectively. (d) A549 cells were treated with 2 mℳ HU. RNA and protein extracts were prepared at the indicated times and miR-29a and Cdc7 levels were determined by RT–qPCR and western blot respectively.

Figure 4

Cdc7/Dbf4 regulation, checkpoint signaling and survival are impaired in miR-29a-expressing cells upon BPDE-induced DNA damage. (a) miR-ve and miR-29a-expressing A549 cells were treated with 100 nM BPDE. At different times after BPDE treatment, CSK extracts were prepared and analyzed by western blot for the specific proteins noted. (b) A549 cells were transfected with miR-ve or miR-29a and 24 h later were treated with different doses of BPDE (50, 100, 200 or 600 nℳ) and colony forming assays performed. n=3, ***P=0.001, **P=0.03.

Figure 5

miRNA-resistant Cdc7/Dbf4 reverse miR-29a-induced sensitization to BPDE. A549 cells were co-transfected with HA-Dbf4 and FLAG-Cdc7 expression vectors or with pcDNA 3.1 empty vector control and subsequently transfected with miR-ve or miR-29a. Twenty-four hours later, cells were (a) harvested for Western blot analysis or (b) treated with 100 nM BPDE and analyzed for clonogenic survival. n=4, **P=0.019.